猪圆环病毒2型免疫过氧化物酶单层细胞试验抗体检测试剂盒的研制及应用

本研究建立了一种免疫过氧化物酶单层细胞试验（Immunoperoxidase monolayer assay,IPMA）,用于猪圆环病毒2型血清抗体检测,通过对IPMA反应条件的优化,组装了诊断试剂盒。研究结果表明,用IPMA检测猪圆环病毒2型人工感染猪血清,于感染后3周抗体阳转,第3周～10周抗体阳性检出率为92.8%（52/56）,对照组猪血清抗体检测均为阴性（33/33）。试剂盒在-20℃稳定保存18个月与其他几种猪病毒参考血清无交叉反应,与用重组蛋白抗原建立的rcELISA符合率为89.2%。对来自黑龙江、吉林、河北、上海、内蒙古、云南、江西等地猪场健康成年猪血清480份和发病猪血清424份进行了检测,抗体检出率分别为91.7%和79.2%,表明我国猪群中猪圆环病毒2型污染相当严重。该试剂盒的研制为我国PCV2流行病学调查和疫苗免疫效果的评价提供了技术手段。     

2009年我国猪群中猪圆环病毒2型感染情况调查

为了解我国猪群中猪圆环病毒2型（Porcine circovirustype2，PCV2）的感染情况，分析感染与猪年龄的关系，本试验于2009年在我国28个省市的71个中小规模场、22个屠宰场和62家散养户采集了2905份猪血清样品，用PCR法对PCV2进行检测。结果显示，检出阳性血清429份，阳性率为14．8％。在被调查的场／P中，86．4％屠宰场、52．1％中小规模场和58．1％散养户为PCV2感染阳性，其中48．6％的PCV2阳性中小规模场的群阳性率低于10％。分析中小规模场PCV2感染情况和猪年龄之间的关系发现，在5个年龄组中，2～4周龄组未检出PCV2感染，5周龄以上各组均检出PCV2感染，其中11～14周龄组和15～26周龄组阳性率较高，高于5～7周龄组和8～10周龄组。     

华南地区猪圆环病毒和猪繁殖与呼吸综合征病毒检测分析

为了解华南地区猪圆环病毒及猪繁殖与呼吸综合征病毒的最新流行情况,采集了华南地区11个规模猪场各饲养阶段猪血清807份,用套式PCR(nPCR)检测猪圆环病毒1型(PCV-1)和猪圆环病毒2型(PCV-2);采集了若干猪场2010年1月至2011年8月期间有咳嗽、喘气、消瘦及疑似PDNS等临床症状的猪血清312份,以及无临床症状猪血清104份,以nPCR检测PCV-2,以一步法反转录PCR(RT-PCR)检测猪繁殖与呼吸综合征病毒。结果发现,所调查的11个规模猪场中只有5个检出PCV-1,所有猪场均检出PCV-2。PCV-2阳性率为30.61%,而PCV-1阳性率仅为4.21%;经产母猪和7周龄以上保育猪PCV阳性率最高;有临床症状的猪血清PCV-2阳性率为58.65%,PRRSV阳性率为37.82%,PCV-2阳性猪群中有39.89%的猪同时感染PRRSV;有症状猪群7月～9月的PCV-2感染率最高,而1月～3月最低;无临床症状猪血清PCV-2阳性率为27.9%,PRRSV阳性率为0.96%,PCV-2与PRRSV无混合感染。证明PCV尤其是PCV-2在华南地区仍广泛传播并流行,而且PCV-2与PRRSV混合感染致病情况较多。PCV-2的感染率与季节有一定的相关性,种猪带毒情况严重。     

Development and application of a competitive enzyme-linked immunosorbent assay for the detection of serum antibodies to porcine circovirus type 2.

We report the development of a competitive enzyme-linked immunosorbent assay (c-ELISA) for the detection of antibodies to porcine circovirus type 2 (PCV2), the agent associated with the recently described postweaning multisystemic wasting syndrome in pigs. At present, no method has been published describing a c-ELISA for the detection of antibodies to PCV2, and currently employed tests are impractical for use in some laboratories. The assay described here uses a cell culture isolate of porcine circovirus type 2 as antigen and a PCV2-specific monoclonal antibody as the competing reagent. Evaluation of the ELISA was performed by comparison with results obtained using an indirect immunofluorescent test on 484 sera from pig herds in the United Kingdom, Canada, France, and the USA and serial bleeds from pigs experimentally infected with porcine circoviruses. The sensitivity and specificity of the ELISA were determined as 99.58% and 97.14%, respectively, at 2 standard deviations (SD) from the mean or 95.81% and 100% at 3 SD from the mean. Using this ELISA, a serologic survey of 461 sera collected from commercial pig herds in Northern Ireland between 1973 and 1999 was undertaken. Analysis of the results of this survey demonstrated that the number of ELISA-positive sera detected in an individual year during this period ranged from 55% to 100%. This c-ELISA has applications for large-scale rapid diagnosis of PCV2 infection in pig populations worldwide and for immunoscreening of sera from other species for antibodies to PCV2.     

Retrospective study on porcine circovirus type 2 infection in pigs from 1985 to 1997 in Spain

A retrospective survey was performed to detect lesions of Postweaning multisystemic wasting syndrome (PMWS) and nucleic acid of porcine circovirus type 2 (PCV2) in archived formalin-fixed, paraffin-embedded tissues from 189 pigs, and antibodies to this virus in sera of 388 pigs from the Spanish livestock between the years 1985 and 1997. PCV2 nucleic acid was detected by in situ hybridization (ISH) in tissues from 78 of 189 (41.3%) examined pigs. Variable amount of viral genome was detected in association with slight to severe microscopic lymphoid lesions consisting of lymphocyte depletion and histiocytic infiltration. The first positive case of PMWS with typical lesions and ISH positive corresponded to a pig necropsied in 1986. Two hundred and eighty-two of 388 (72.7%) sera were positive by immunoperoxidase monolayer assay. Serological and pathological data of the present study indicate that PCV2 was a enzootic infection in Spain since 1985, suggesting that the introduction of this virus in the livestock occurred previously.     

猪圆环病毒2型细胞培养适应毒株的培育和鉴定

从临床表现为仔猪断奶后多系统衰竭综合征（PMWS）淋巴组织病料，经聚合酶链式反应（PCR）证实为猪圆环病毒2型（PCV2）感染，采用无污染的猪肾细胞系（PK15）分离培养，并连续传代培育成一株细胞培养适应毒，命名为PCV2／LG株。分离毒株经细胞培养，于第25代后毒价显著升高，于第35代毒价可达10^5.6TCID 50/mL。采用免疫过氧化物酶单层细胞染色法（IPMA）、免疫电镜技术、分子克隆及核酸序列分析等鉴定表明，分离株感染细胞后病毒抗原主要分布在细胞核及细胞质中；病毒感染的阳性细胞呈散在分布，阳性细胞数可达50％以上；免疫电镜观察到与PCV2特异抗体结合形成的病毒免疫复合物呈实心小颗粒样粒子团，病毒粒子直径约为17nm；病毒抗原基因组由1768个核苷酸组成，与GenBank登录的8个PCV2基因组序列同源性达96．2％以上。用2mL的病毒细胞培养物（10^5.6TCID 50/mL）接种30日龄PCV2抗体阴性仔猪3头，可引起典型PMWS临床症状。本研究为进一步开展该病毒的致病性、疫苗免疫、诊断及分子生物学等研究奠定了基础。     

Infectivity of porcine circovirus 1 and circovirus 2 in primary porcine hepatocyte and kidney cell cultures

Infectivity of porcine circovirus (PCV) 1 and PCV2 was examined in primary porcine hepatocyte culture by comparing that of PCV in primary kidney cell culture. The virus titer of PCV2-infected hepatocyte cultures was higher than that of the PCV1-infected hepatocyte cultures and the PCV-infected kidney cell cultures. The number of virus-positive cells was most abundant in PCV2-infected hepatocyte cultures as determined by immunohistochemistry and/or in situ hybridization. The results of our data suggest that PCV2 preferably infects cultured hepatocytes as observed in the liver of pigs with postweaning multisystemic wasting syndrome.     

猪圆环病毒Ⅱ型Rep基因在PK15细胞中的表达及特性

为研究猪圆环病毒Ⅱ型(PCV2)Rep基因在PK15细胞中的表达特性，通过PCR方法克隆了PCV2杭州株(HZ0201)Rep基因全长945bp片段，与真核表达栽体pCI—neo构建为重组质粒pCI-PCV2-Rep。pCI-PCV2-Rep质粒转染PK15细胞后48h，通过RT-PCR可检测到PCV2 Rep mRNA的转录；用猪PCV2多抗血清作间接免疫荧光试验，可检测到Rep基因表达产物。在表达量低的细胞中，PCV2 Rep蛋白主要位于PK15的细胞浆，在表达量高的细胞中，细胞浆和细胞核中均含有大量的Rep蛋白，表明Rep对PK15细胞的细胞浆和细胞核的亲嗜性没有明显差别。     

Fatal bronchopneumonia in a Metastrongylus elongatus and Porcine circovirus type 2 co-infected pig

Porcine circovirus type 2 (PCV2) infection is distributed worldwide and PCV2-associated disease (PCVAD) is considered among the most economically relevant ones to the global swine industry. PCV2 is known to play a causal role in the porcine respiratory disease complex, usually in close association with a large plethora of other biologic agents. We describe herein a case of fatal parasitic bronchopneumonia by Metastrongylus elongatus in a PCV2-infected pig. Metastrongylosis may still represent a major concern for outdoor herds. Our recent experience suggests that a concurrent PCVAD condition may trigger metastrongylosis, which may subsequently result, at its turn, in severe, sometimes fatal, pulmonary disease.     

Isolation and characterization of porcine circovirus type 2 from pigs showing signs of post‐weaning multisystemic wasting syndrome in the Netherlands

Summary

Pigs with wasting syndrome were examined for macroscopic and histopathological lesions, and for porcine circovirus (PCV). Histopathological lesions were comparable to those previously documented for post‐weaning multisystemic wasting syndrome (PMWS). In addition, in seven out of ten examined PMWS‐affected pigs focal‐to‐slight mononuclear meningitis and focal cerebral mononuclear infiltrates (4 out of 10) were observed. A virus was isolated from organs and sera from pigs showing wasting syndrome. An immunoperoxidase monolayer assay and an indirect immunofluorescence assay were performed on the infected PK‐15 and Dulac cell cultures, respectively, and both assays indicated the presence of PCV type 2 (PCV2). The nested‐polymerase chain reaction (nPCR) technique, based on the use of PCV2 specific oligonucleotides, revealed specific amplified products of 481 bp. Nucleotide sequence analysis of the entire genome of the Dutch PCV isolate 24657 NL showed a homology with known nucleotide sequences of porcine PCV type 1 (PCV1) and PCV2 isolates of 77.1% and >96%, respectively. This is the first report of the isolation and characterization of PCV2 in PMWS‐affected pigs in the Netherlands.     

Characterization of porcine circovirus type 2 in Taiwan

In an effort to understand the genetic diversity of porcine circovirus type 2 (PCV2) and the prevalence of PCV2 infection in Taiwanese herds, we have sequenced the complete genomes from PCV2-infected specimens and individually measured the antibody titer against PCV2 from pigs reared in Taiwan between the years 2000 and 2002. A total of 623 specimens originating from pigs displaying varied clinical signs were screened with the polymerase chain reaction (PCR). Results showed that 309 pigs (49.6%) tested positive for PCV2. Eight of the positive specimens were used for the amplification of the complete viral genome. Sequence comparison of the complete genomes indicated that the 8 Taiwanese PCV2 isolates shared 95-99% similarity. Phylogenetic analysis of all 40 PCV2 isolates from North America, Europe, Asia and Taiwan revealed that those isolates were grouped together in one large group containing two minor subgroups. The Taiwanese PCV2 isolates were classified into the two minor subgroups. The prevalence of serum antibodies to PCV2 in pigs was investigated, and results showed that approximately 83.5% of the pigs in Taiwan were seropositive. Finishing pigs possess the highest titers of antibodies, while 9-week-old pigs contained the lowest titers for specific antibodies. Our results suggest that PCV2 infections have become common in Taiwanese pig farms.     

Porcine circovirus type 2 viremia and seroconversion in pigs from a farm affected by postweaning multisystemic wasting syndrome

The aim of the study was to assess the usefulness of real-time PCR and serological methods as indicators of postweaning multisystemic wasting syndrome (PMWS) occurrence. Significantly higher level of porcine circovirus type 2 (PCV2) viral load in serum and significantly lower titre of specific antibodies in PMWS-affected pigs indicated that combination of quantitative PCR and serological methods may support diagnosis of PMWS.     

Reproduction of postweaning multisystemic wasting syndrome (PMWS) in immunostimulated and non-immunostimulated 3-week-old piglets experimentally infected with porcine circovirus type 2 (PCV2)

Postweaning multisystemic wasting syndrome (PMWS) in swine is causally associated with the newly recognised pathogen, porcine circovirus type 2 (PCV2). In this study, 3-week-old SPF PCV2-seronegative piglets were inoculated intranasally with PCV2. The effect of immunostimulation on the induction of PMWS was investigated by immunisation with keyhole limpet hemocyanin (KLH) emulsified in incomplete Freunds adjuvant. The study was terminated 5 weeks after inoculation. While disease was not observed in the age-matched controls, two out of five non-immunised PCV2-infected piglets died on postinoculation day (PID) 21, and one was euthanized on PID 25 in moribund condition. These animals had appeared lethargic with persistent fever from PID 12 onwards. The euthanized pig appeared smaller than littermates and suffered from jaundice. At postmortem examination, gastric ulceration, icterus, and liver and thymus atrophy were observed. Furthermore, histological lesions of degenerating hepatocytes and hepatitis in combination with lymphoid depletion and syncytial cells in lymph nodes were consistent with the diagnosis of PMWS. One out of five immunostimulated PCV2-infected piglets was euthanized on PID 22 with convulsions after a period with wasting. This pig was lethargic from PID 14 onwards with persistent fever from PID 8 and transient dyspnoea. No differences in clinical signs, gross pathologic or histological findings were observed for the remaining non-immunostimulated and immunostimulated PCV2-infected piglets. All 10 PCV2-inoculated piglets seroconverted to PCV2 within 14 days after inoculation. By virus isolation, quantitative polymerase chain reaction (Q-PCR), and immunostaining of cryostat sections, it was demonstrated that lymphoid tissue contained abundant PCV2 antigen. Viral DNA load in serum samples was assessed by Q-PCR. All four PMWS-affected piglets had high levels of PCV2 DNA in serum, suggesting that there was a correlation between high levels of viral DNA in serum and the development of PMWS. In conclusion, infection with PCV2 caused PMWS in SPF piglets, however, the immunostimulation did not seem to play a critical role.     

Inhibition of porcine circovirus type 1 and type 2 production in PK-15 cells by small interfering RNAs targeting the Rep gene

Porcine circovirus type 1 (PCV1) and type 2 (PCV2) are two genotypes of porcine circovirus. Both of them are presumed to be widespread in the swine population. Currently, there is no specific treatment for their infections. RNA interference (RNAi) is a sequence-specific RNA degradation mechanism mediated by small interfering RNA (siRNA), which represents a possible therapeutic application for the treatment of viral infections. In this study, three siRNA expression plasmids (pS-RepA, pS-RepB and pS-RepC) were generated to target three different coding regions of the Rep protein (Rep) of PCV. These siRNAs were used to inhibit PCV production in a porcine kidney cell line, PK-15 cells. Our results revealed that Rep gene expression was inhibited by pS-RepA, pS-RepB and pS-RepC to different degrees. Moreover, our study also showed that the production of PCV1 and PCV2 was reduced by these siRNAs. pS-RepC, which targets the middle region of Rep gene, proved to be the most efficient siRNA for inhibition of Rep expression and viral production. Taken together, our data suggest that RNAi could be investigated as a potential treatment for PCV infection.     

Survey of porcine circovirus 2 and postweaning multisystemic wasting syndrome in New South Wales piggeries

OBJECTIVE: To determine if postweaning multisystemic wasting syndrome (PMWS) is occurring in the New South Wales pig population and to determine the current and past seroprevalence of porcine circovirus 2 (PCV2). DESIGN: Pig veterinarians were contacted seeking submission of tissues from animals with clinical signs suggestive of PMWS. Samples were also accepted from suspected cases of porcine dermatitis and nephropathy syndrome (PDNS). Serological studies were also undertaken on archival sera and sera submitted during the study. PROCEDURE: Histopathological examination was undertaken on all tissues submitted. The presence of PCV2 was determined by immunohistochemistry. Sera were tested for PCV2 using a commercial enzyme linked immunosorbent assay kit modified for testing of serum samples. RESULTS: No cases of PMWS were identified during the study. Four cases of PDNS were identified. PCV2 antibody was detected in 80% of archival sera from 1995 and 75.8% from 2001. Seroprevalence in samples tested during 2002-2003 was 87.8%. PCV2 was isolated from tissues of a case of PDNS. CONCLUSION: PCV2 is widespread in the New South Wales pig population and has been since at least 1995. This study describes the first isolation of an Australian PCV2. No cases of PMWS were identified in New South Wales.     

猪圆环病毒2型胶体金抗体检测方法的建立

为建立检测猪圆环病毒2型(PCV2)胶体金检测方法,本研究对SPA蛋白进行胶体金标记,并喷涂于玻璃纤维上制备金标垫,分别以重组PCV2 ORF2蛋白和猪IgG作为检测线和质控线,制作PCV2抗体检测胶体金免疫层析试纸条。检测结果表明,试纸条操作简单,肉眼于10 min内可以判定结果,对PCV2免疫血清具有高度特异性,与猪其它病毒免疫血清无交叉反应,检测灵敏度与ELISA相近。试纸条在室温保存6个月,其特异性及灵敏度无明显变化。对临床采集的324份血清样品进行检测,结果与ELISA试剂盒总符合率为98.77%。表明本研究建立的PCV2抗体免疫层析检测方法具有特异、敏感、稳定、操作简单快捷等特点,适合于PCV2抗体的现场检测。     

Detection of porcine circovirus type 1 in commercial pig vaccines using polymerase chain reaction

The absence of extraneous viruses is a requirement in the quality control of vaccines for veterinary use in the European Pharmacopoeia. A polymerase chain reaction (PCR) assay for the detection of porcine circovirus type 1 (PCV1) and type 2 (PCV2) was evaluated in 18 commercial porcine vaccines. Since vaccine components may contain PCR enhancers or inhibitors, 13 of the studied vaccines (used as diluents) were subsequently spiked with different dilutions of PCV2 and tested by PCR. Although PCV2 DNA was not detected in any of the vaccines tested, PCV1 was detected in 2/18 vaccines (11%). Eleven out of 13 PCV2 spiked vaccines showed a positive PCR result. The lack of amplification observed in two spiked vaccines suggested that use of the PCR assay to detect PCV2 could depend on vaccine composition. The results of this exploratory study have demonstrated that PCR is a rapid and fairly sensitive method for the detection of porcine circoviruses as extraneous agents in vaccine products and can be used in the quality control of pig vaccines. The study has also indicated the need for optimising the sensitivity of PCR methods for PCV genome detection in vaccine products.     

Development of probes to differentiate porcine circovirus types 1 and 2 in vitro by in situ hybridization

Porcine circovirus type 1 (PCV1), a PK-15 cell line contaminant, and porcine circovirus type 2 (PCV2), associated with post-weaning multisystemic wasting syndrome (PMWS), are genetically and antigenically related. Several techniques have been developed to detect PCV, including in situ hybridization (ISH). Previously reported probes used for ISH may hybridize with both PCV1 and PCV2 nucleic acids. We attempted to produce probes for ISH that can detect and differentiate PCV2 from PCV1 in PCV-infected cells. Riboprobes were synthesized from the sense and antisense strands of both open reading frames 1 and 2 (ORF1 and ORF2) of PCV2. At 42 and 58 degrees C, the ORF1 antisense probe hybridized with nucleic acid from both PCV1- and PCV2-infected cells. At 58 degrees C, the ORF2 antisense probe hybridized with PCV2 nucleic acid but not with PCV1 nucleic acid. The ORF1 and ORF2 sense probes bound only with PCV2 nucleic acid. Both antisense strand probes produced stronger signals than the sense strand probes. The results showed that the PCV2 ORF1 antisense probe is the most likely probe to detect both PCV types while the ORF2 antisense probe is capable of discriminating between PCV1 and PCV2.     

Distribution and characterization of IL-10-secreting cells in lymphoid tissues of PCV2-infected pigs

Distribution and characterization of interlukin-10 (IL-10)-secreting cells in lymphoid tissues of pigs naturally infected with porcine circovirus type 2 (PCV2) were evaluated in accordance with PCV2 antigen detection. After screening a total of 56 pigs showing the symptoms of postweaning multisystemic wasting syndrome (PMWS), 15 pigs were PCV2 positive and 5 pigs, which showed stronger positive signals over multiples tissues were further investigated. This study showed that in PCV2-infected lymphoid tissues, particularly mandibular lymph node, spleen and tonsil, IL-10 expression was mainly localized in T-cell rich areas but rarely in B cell rich areas. IL-10 was highly expressed in bystander cells but rarely in PCV2-infected cells. Elevated IL-10 expression was predominantly associated with T cells, but rarely with B cells or with macrophages. The results of this study provide evidence for the role of IL-10 in chronic PCV2 infection and its relation to PCV2 antigen in affected tissues. Constantly elevated levels of IL-10 lead to immunosuppression in persistent and chronic viral infections. The increased IL-10 expression observed in PCV2 infection in this study suggests that IL-10-mediated immunosuppression may play an important role in the pathogenesis and maintenance of naturally occurring PCV2 infection.     

Experimental inoculation of porcine circoviruses type 1 (PCV1) and type 2 (PCV2) in rabbits and mice

The objective of this work was to investigate the susceptibility of rabbits and mice experimentally inoculated with porcine circoviruses type 1 (PCV1) and type 2 (PCV2) to infection and development of disease and/or lesions. Forty six New Zealand rabbits and 50 ICR-CDI mice were both divided into two groups comprising PCVI and PCV2 inoculated animals, and a third group inoculated with non-infected cell culture medium. Rabbits were inoculated intranasally while mice were inoculated intraperitoneally. Clinical signs and body weights were recorded at the start of the experiment and at necropsy. Animals were bled, euthanised and necropsied at days 0, 3, 7, 10, 14 and 20 post-inoculation and samples were collected for histopathological, serological, in situ hybridisation and PCR analysis. No clinical signs or gross and microscopic lesions compatible with PCV2 infections such as those seen in pigs were observed. No presence of PCV2 nucleic acid was detected in rabbits and mice by in situ hybridisation. Only one mouse inoculated with PCV1 seroconverted on day 20 P1. PCV1 and PCV2 genome was detected in serum by PCR in mice inoculated with each porcine circovirus, while rabbits were negative for both viral types. These studies indicated that porcine circoviruses did not cause any disease or microscopic lesions in inoculated rabbits and mice during the experimental period. However, intraperitoneally inoculated mice might have harboured PCV2 in circulation without evidence of viral replication.